The objective of this Project is to develop selective genetic and pharmacological tools to perturb the major pathways for endocannabinoid (EC) metabolism. These pathways can roughly be divided into four separate groups: 1) anandamide (AEA) biosynthesis, 2) AEA degradation, 3) 2-arachidonoyl (2-AG) biosynthesis, and 4) 2-AG degradation. Among these processes, AEA degradation is the most mature in terms of our scientific understanding and availability of research tools. Studies over the past decade using knockout mice and selective inhibitors have demonstrated that the integral membrane protein fatty acid amide hydrolase (FAAH) is the primary enzyme responsible for terminating AEA signaling in the central nervous system (CMS). In contrast, our understanding of the enzymes involved in the other major branches of EC metabolism is much less complete. Although candidate enzymes have been identified in these pathways, their actual contribution to EC metabolism in vivo remains largely unknown due to a dearth of selective research tools to probe their function in living systems. We plan to address this crucial problem by creating knockout mouse models and selective chemical inhibitors for key enzymes implicated in EC metabolism. These enzymes include: 1) the putative AEA biosynthetic enzymes, a/b-hydrolase 4 (Abh4) and glycerophosphodiesterase-1 (GDE-1), 2) the putative 2-AG degradative enzyme, monoacylgycerol lipase (MAGL), and 3) the putative 2-AG biosynthetic enzyme diacylgycerol lipase-alpha (DAGL-a). Importantly, these research efforts will be bolstered by a set of innovative technologies that will ensure efficient generation of high-quality research tools for rapid dissemination among the Program Project team and larger EC research community. Collectively, the studies described in this Project will generate the requisite genetic and pharmacological tools to systematically delineate the key enzymes involved in all major branches of EC metabolism. The following three major hypotheses will be tested: 1) AEA is biosynthesized by a pathway involving the sequential action of Abhd4 and GDE-1; 2) 2-AG catabolism is regulated by multiple enzymes in vivo, with a significant contribution attributable to MAGL; 3) 2-AG biosynthesis in the mature brain is predominantly controlled by DAGL-a.